Vaccine against acinetobacter baumannii based on cellular components deficient in lipopolysaccharide

ABSTRACT

The invention refers to a composition comprising inactivated cells deficient in LPS from the genus  Acinetobacter  and/or outer membrane vesicles form the same and their use for the manufacture of a medicament, preferably a vaccine, for the prevention of diseases produced by organisms of the genus  Acinetobacter.

RELATED APPLICATIONS

This application is continuation of U.S. application Ser. No.15/878,491, filed Jan. 24, 2018 and now pending, which is a divisionalof U.S. application Ser. No. 15/308,899, filed Nov. 4, 2016 and nowabandoned, which is a national stage filing under 35 U.S.C. § 371 ofInternational Application No. PCT/EP2015/059870, filed May 5, 2015, theentire contents of each of which is incorporated by reference herein inits entirety.

REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS-WEB

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Sep. 20, 2021, isnamed H007570220US02-SEQ-JRV.txt, and is 223,904 bytes in size.

FIELD OF THE INVENTION

The present invention refers, in general, to the field of pharmacologyand immunology, and, in particular, to a vaccine composition suitablefor the prophylactic treatment of an infection caused by anAcinetobacter baumannii strain in a mammal.

BACKGROUND OF THE INVENTION

The increasing clinical importance of infections caused by multidrugresistant A. baumannii warrants the development of novel approaches forthe prevention and treatment of infections caused by this pathogen. Inthis context, immunization of certain patient populations couldcontribute to reducing the morbidity and mortality caused by thispathogen. Vaccines against Gram-negative bacteria based on inactivatedwhole bacterial cells are highly immunogenic and have been shown toproduce protective immunity against a number of bacterial species.However, the high levels of endotoxin, due to the presence oflipopolysaccharide, in these vaccines complicate their use in humans.Acinetobacter baumannii is a Gram-negative coccabacillus with clinicalimportance in the hospital environment. This organism is highlydistributed in soil and environmental water sources (Baumann, P. 1968.J. Bacteriol. 96, 39-42), and can cause different types of infections asa nosocomial pathogen such as pneumonia, bacteremia, meningitis and skinand soft tissue infection, among others (Garcia-Quintanilla et al.,2013. Curr. Pharm. Biotechnol. In press). This pathogen typicallyinfects patients receiving mechanical ventilation and patientssustaining burn injury (Munoz-Price y Weinstein. 2008. N. Engl. J. Med.358, 1271-1281). However, it has also been isolated in cases ofcommunity-acquired pneumonia (Ho et al., 2009. Chest. 136, 1119-1127;Ong et al., 2009. Respirology 14, 1200-1205) and in military personnelsustaining war-related trauma in Vietnam, Iraq, Kuwait and Afghanistan(Jones et al., 2006. Lancet Infect. Dis. 6, 317-318; Tong, 1972. JAMA219, 1044-1047). The crude mortality rates associated with infection byA. baumannii are between 35% and 70% for nosocomial infections (Vila yPachon, 2008. Expert Opin. Pharmacother. 9, 587-599). Due to the abilityof A. baumannii to acquire resistance to antibiotics, the number ofmultidrug resistant strains has increased dramatically over thepreceding years (Tasbakan et al., 2009. Mikrobiyol Bul. 43, 61-70;Valencia et al., 2009. Infect. Control Hosp. Epidemiol. 30: 257-263).The appearance of these highly resistant strains has complicated theclinical Management of infections caused by A. baumannii. In thiscontext, the development of a vaccine against A. baumannii could reducethe morbidity and mortality caused by this pathogen (Pachón y McConnell,2014. Vaccine, In press).

The experimental vaccines that have been described for A. baumannii canbe classified into two broad groups, vaccines consisting of a singlepurified antigen, and multicomponent vaccines. Within the first group,the outer membrane protein OmpA (Luo et al., 2012. PLoS One 7, e29446),the biofilm associated protein Bap (Fattahian et al., 2011. Microb.Pathog. 51, 402-406), the membrane transporter Ata (Bentancor et al.,2012. Infect. Immun. 80, 3381-3388), and the surface polysaccharidepoly-N-acetyl-β-(1-6)-glucosamine (Bentancor et al., 2012. Infect Immun80, 651-656) have been described as good candidates due to theircapacity to elicit a specific immune response. However, the experimentstesting survival after active immunization have only demonstrated thatOmpA provides partial protection, and that the expression of Bap has notbeen clearly demonstrated in strains that do no form biofilm. Theapproaches that employ multicomponent vaccines include outer membranecomplexes (McConnell et al., 2011. Infect. Immun. 79, 518-526), outermembrane vesicles (outer membrane vesicles; McConnell et al., 2011.Vaccine 29, 5705-5710) and inactivated whole cells (McConnell y Pachon,2010. Vaccine 29: 1-5). Each of these vaccines induces a strong immuneresponse and is able to elicit high levels of protection againstinfection in a murine model using the ATCC 19606 strain and clinicalisolates. However, in spite of these promising results, the use of thesevaccines in humans is complicated in view of the high levels ofendotoxin in these vaccines due to the high amounts oflipopolysaccharide (LPS) present in these preparations.

LPS is formed by the O antigen, a core polysaccharide and lipid A, whichis responsible for the endotoxin activity of LPS. The first studies thatemployed Escherichia coli demonstrated that the production of LPS wasessential for bacterial viability (Raetz, 1990. Annu. Rev Biochem. 59,129-170). However, subsequent work demonstrated that certain bacterialspecies, such as Neisseria meningitidis y Moraxella catarrhalis, wereviable after mutation of the genes encoding enzymes involved in LPSbiosynthesis, resulting in strains completely lacking LPS (Peng et al.,2005. Infect. Immun. 73, 7569-7577; Steeghs et al., 1999. Infect. Immun.67, 4988-4993). A recent study demonstrated that A. baumannii canacquire resistance to the antibiotic colistin through mutation of thegenes involved in LPS biosynthesis IpxA, IpxC and IpxD (Moffatt et al.,2010. Antimicrob. Agents Chemother. 54, 4971-4977), living rise tostrains completely deficient in LPS. This results indicate that A.baumannii is also viable in the absence of LPS, raising the possibilityof developing vaccines based on these strains.

Outer membrane vesicles (OMVs) are vesicles derived from the bacterialouter membrane that are secreted from numerous Gram-negative bacteria(Kulp et al., 2010. Annu. Rev. Microbiol. 64, 163-184). OMVs arespherical vesicles of approximately 20-200 nm that are composed of outermembrane proteins, periplasmic proteins and LPS (Kuehn et al., 2005.Genes Dev. 19, 2645-2655; Mashburn et al., 2005. Nature. 437, 422-425).Secreted OMVs have been shown to participate in the detection of quorumsensing, the transport of virulence factors and the transfer of genes,indicating that they play a role in bacterial patogenesis. It has alsobeen demonstrated that OMVs can deliver proteins to the interior of hostcells through fusion with lipid rafts, suggesting that OMVs can be usedto transport bacterial products over large distances (Kesty et al.,2004. Embo J. 23, 4538-4549). A recent study by Kwon et al demonstratedthat a clinical isolate of A. baumannii secreted OMVs during growth invitro (Kwon et al., 2009. FEMS Microbiol. Lett. 297, 150-156). Aproteomic analysis of the OMVs demonstrated that they contain multiplevirulence factors and immunomodulating proteins, suggesting that OMVsplay an important role in the pathogenesis of A. baumannii.

Vaccines based on OMVs have been developed for various Gram-negativebacteria including Neisseria meningitidis, Helicobacter pylori, andVibrio cholerae (Bjune et al., 1991. NIPH Ann. 14, 125-130; Keenan etal., 2003. FEMS Immunol. Med. Microbiol. 36, 199-205; Bishop et al.,2012. J. Infect. Dis. 205, 412-21). Immunization with OMVs has beenshown to induce antibodies against multiple bacterial antigens, and thecapacity to provide protective immunity in animal models of infection.In addition, the OMVs isolated form N. meningitidis serogroup B havebeen shown to be safe and immunogenic in humans, and have been used tocontrol an outbreak of meningococcus meningitis in New Zealand (Noklebyet al., 2007. Vaccine. 25, 3080-84).

The infections caused by A. baumannii often occur in outbreaks caused bya single clone. For this reason, a vaccine based on OMVs could be moreeffective if the OMVs are isolated form the causative clone. Thepurification of OMVs from bacterial cultures is rapid and simple,requiring only filtering of the culture supernatant and concentration ofthe OMVs.

BRIEF DESCRIPTION OF THE INVENTION

Aspect A: This aspect of the invention refers to a pharmaceuticalcomposition, preferably a vaccine composition, suitable for theprophylactic treatment (before infection) of an infection caused by anAcinetobacter baumannii strain in a mammal, which comprises:

-   -   a. an Acinetobacter baumannii strain whole cell deficient in        lipopolysaccharide (LPS) characterized by the partial or        complete inactivation of one or various cellular nucleic acid        molecules that encode endogenous LPS; and/or    -   b. an outer membrane vesicle (OMV) derived from an Acinetobacter        baumannii strain deficient in lipopolysaccharide (LPS) as        defined in paragraph a) above.

In a prefer embodiment of this aspect of the invention, theAcinetobacter baumannii strain deficient in lipopolysaccharide (LPS) ischaracterized by the partial or complete inactivation of the genesselected from the list consisting of IpxA, IpxB and/or IpxC.

In another prefer embodiment of this aspect of the invention or of anyof its preferred embodiments, the pharmaceutical composition, preferablythe vaccine composition, further comprises a, preferably recombinant,polypeptide selected from the list consisting of:

-   -   a. Aminoacid sequence SEQ ID No 27 (putative ferric siderophore        receptor (A. baumannii ATCC 17978; accession number        YP_001084684)) or a fragment thereof, wherein the term fragment        is understood herein as biologically active fragments selected        from the list consisting of SEQ ID No 1 to SEQ ID NO 11 or any        combination thereof, or an amino acid sequence having at least        85% identity with any of sequences SEQ ID NO 1 to SEQ ID No 11;        and/or    -   b. Amino acid sequence SEQ ID No 28 (putative ferric hydroximate        siderophore receptor (A. baumannii ATCC 17978; accession number        YP_001084696)) or a fragment thereof, wherein the term fragment        is understood herein as biologically active fragments selected        from the list consisting of SEQ ID NO 12 to SEQ ID NO 23 or any        combination thereof, or sequences that have at least 85%        identity with the amino acids SEQ ID NO 12 to SEC ID NO 23.

In another prefer embodiment of this aspect of the invention or of anyof its preferred embodiments, the pharmaceutical composition, preferablythe vaccine composition, further comprises a purified outer membraneprotein sequence of A. baumannii selected from the list consisting of:SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO:33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ IDNO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46; SEQ ID NO: 47;SEQ ID NO: 48; SEQ ID NO: 49; SEQ ID NO: 50; SEQ ID NO: 51; SEQ ID NO:52; SEQ ID NO: 53; SEQ ID NO: 54; SEQ ID NO: 55; SEQ ID NO: 56; SEQ IDNO: 57; SEQ ID NO: 58; SEQ ID NO: 59; SEQ ID NO: 60; SEQ ID NO: 61; SEQID NO: 62; SEQ ID NO: 63; SEQ ID NO: 64; SEQ ID NO: 65; SEQ ID NO: 66;SEQ ID NO: 67; SEQ ID NO: 68; SEQ ID NO: 69; SEQ ID NO: 70; SEQ ID NO:71; SEQ ID NO: 72; SEQ ID NO: 73; SEQ ID NO: 74; SEQ ID NO: 75; SEQ IDNO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID No: 80, SEQID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85,SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, or anycombination thereof.

In yet another prefer embodiment of this aspect of the invention or ofany of its preferred embodiments, the pharmaceutical composition,preferably the vaccine composition, further comprises fusion recombinantpolypeptides sequences SEQ ID NO 24 and/or 25 and/or the amino acidsequence coded by nucleotide sequence SEQ ID NO 26.

In yet another prefer embodiment of this aspect of the invention or ofany of its preferred embodiments, the Acinetobacter baumannii straindeficient in lipopolysaccharide (LPS) comprises or is transformed,transduced or transfected with a nucleotide sequence capable of codingfor any of the amino acid sequences as defined in any of the precedentparagraphs so that such strain is capable of producing the exogenousexpression any of these amino acid sequences.

In yet another prefer embodiment of this aspect of the invention or ofany of its preferred embodiments, said pharmaceutical composition,preferably the vaccine composition, further comprises a vector, such asviral vector, a plasmid or an expression cassette comprising anucleotide sequence capable of coding for any of the amino acidsequences as defined in any of the precedent paragraphs and expressingsaid amino acid sequences.

In yet another prefer embodiment of this aspect of the invention or ofany of its preferred embodiments, the Acinetobacter baumannii straindeficient in lipopolysaccharide (LPS) is inactivated. Preferably, saidstrain or cell is derived from ATCC strain 19606.

Aspect B: This aspect of the invention refers to the pharmaceuticalcomposition, preferably the vaccine composition, of the aspect A of theinvention or of any of its preferred embodiments, for use in theprophylactic treatment or for the active immunization of an infectioncaused by A. baumannii in a mammal, preferably in a human.

Aspect C: This aspect of the invention refers to a vaccine compositioncomprising an antibody (monoclonal or polyclonal) or a fragment thereof,preferably selected from the list consisting of Fab, Fab′, Fab′-SH, Fv,scFv, F(ab′)₂, Vhh, nanobody and diabody, having affinity or bindingaffinity against the Acinetobacter baumannii strain deficient inlipopolysaccharide (LPS) and/or against an outer membrane vesicle (OMV)derived therefrom. In a preferred embodiment, said antibody or fragmentthereof specifically binds the Acinetobacter baumannii strain deficientin lipopolysaccharide (LPS) and/or to the outer membrane vesicle (OMV)derived therefrom.

“Affinity” or “binding affinity” KD are often determined by measurementof the equilibrium association constant (ka) and equilibriumdissociation constant (kd) and calculating the quotient of kd to ka(KD=kd/ka).

The term “specifically binding” means that the antibody binds to the LPSdeficient strain or to the OMV derived therefrom with an affinity KD oflower than or equal to 10″⁶M (monovalent affinity). The antibody mayhave substantially greater affinity for the target antigen compared toother unrelated molecules. The antibody may also have substantiallygreater affinity for the target antigen compared to homologs, e.g. atleast 1 0.5-fold, 2-fold, 5-fold 10-fold, 100-fold, 10″³-fold,10″⁴-fold, 10″⁵-fold, 10″⁶-fold or greater relative affinity for thetarget antigen. Such affinities may be readily determined usingconventional techniques, such as by equilibrium dialysis; byradioimmunoassay using radiolabeled target antigen; or by another methodknown to the skilled artisan. The affinity data may be analyzed, forexample, by the method described in [Kaufman R J, Sharp P A. (1982)Amplification and expression of sequences cotransfected with a modulardihydrofolate reductase complementary dna gene. J Mol Biol.159:601-621].

The term “antibody” is used in the broadest sense and includes fullyassembled antibodies, monoclonal antibodies, polyclonal antibodies,multispecific antibodies (e.g., bispecific antibodies), antibodyfragments that can bind the antigen (e.g., Fab′, F′(ab)2, Fv, singlechain antibodies, diabodies), camelbodies and recombinant peptidescomprising the forgoing as long as they exhibit the desired biologicalactivity.

A “functional fragment” or “antigen-binding antibody fragment” of anantibody/immunoglobulin hereby is defined as a fragment of anantibody/immunoglobulin (e.g., a variable region of an IgG) that retainsthe antigen-binding region. An “antigen-binding region” of an antibodytypically is found in one or more hypervariable region(s) of anantibody, i.e., the CDR-1, -2, and/or -3 regions; however, the variable“framework” regions can also play an important role in antigen binding,such as by providing a scaffold for the CDRs.

The term “hypervariable” region refers to the amino acid residues of thevariable domains VH and VL of an antibody or functional fragment whichare responsible for antigen-binding.

Nonlimiting examples of antibody fragments include Fab, Fab′, F(ab′)2,Fv, domain antibody (dAb), complementarity determining region (CDR)fragments, single-chain antibodies (scFv), single chain antibodyfragments, diabodies, triabodies, tetrabodies, minibodies, linearantibodies [Johnson G, Wu T T. (2000) Kabat database and itsapplications: 30 years after the first variability plot. Nucleic AcidsRes. 28:214-218]; chelating recombinant antibodies, tribodies orbibodies, intrabodies, nanobodies, small modular immunopharmaceuticals(SMI Ps), an antigen-binding-domain immunoglobulin fusion protein, acamelized antibody, a VHH containing antibody, or muteins or derivativesthereof, and polypeptides that contain at least a portion of animmunoglobulin that is sufficient to confer specific antigen binding tothe polypeptide, such as a CDR sequence, as long as the antibody retainsthe desired biological activity; and multispecific antibodies formedfrom antibody fragments [Chothia C, Lesk A M. (1987) Canonicalstructures for the hypervariable regions of immunoglobulins. J Mol Biol.196:901-917; Zapata G, Ridgway J B, Mordenti J, Osaka G, Wong W L,Bennett G L, Carter P. (1995) Engineering linear F(ab′)2 fragments forefficient production in Escherichia coli and enhanced antiproliferativeactivity. Protein Eng. 8:1057-1062].

In a preferred embodiment of aspect C of the invention, thepharmaceutical or vaccine composition is obtained or obtainable afterimmunizing a mammal with the vaccine composition as defined in the firstaspect of the invention.

Aspect D: This aspect of the invention refers to the vaccine compositionas defined in aspect C of the invention, for use in the therapeutictreatment (after the infection), or in the passive immunization, of aninfection caused by A. baumannii in a mammal, preferably in a human.

Aspect E: This aspect of the invention refers to an Acinetobacterbaumannii strain deficient in lipopolysaccharide (LPS), transformed,transduced or transfected with a nucleotide sequence capable of codingfor any of the amino acid sequences as defined in aspect A of theinvention so that such strain is capable of producing the exogenousexpression any of said amino acid sequences. Preferably, said A.baumannii deficient strain is use as a medicament.

Aspect F: This aspect of the invention refers to a method for theproduction of antibodies or fragments thereof, preferably selected fromthe list consisting of Fab, Fab′, Fab′-SH, Fv, scFv, F(ab′)2, Vhh,nanobody and diabody, which comprises:

-   -   a. Selecting an antibody or a fragment thereof, preferably from        an antibody library;    -   b. Using an Acinetobacter baumannii strain deficient in        lipopolysaccharide (LPS) and/or an outer membrane vesicle (OMV)        derived therefrom, as an antigenic target;    -   c. Selecting those antibodies or fragments thereof having        affinity or binding affinity or capable of specifically binding        such strain and/or OMV; and    -   d. Producing those antibodies or fragments thereof identified in        step c) above

It is noted, that Serotype specific variability in A. baumannii relieson variations in epitopes of the LPS component. By eliminating the LPScomponent according to the method as detailed in aspect F above, wefocus the antibody raising to antibodies against less variable epitopesof the bacterial surface, not related to the LPS component. Theseepitopes are conserved among A. baumannii strains or individual isolatesbelonging to all A. baumannii international clones used in A. baumanniiclassification. This method thus provides the tools for a universaltreatment against A. baumannii infections independently of the origin ofthe strain(s) responsible for the host infection.

Aspect G: This aspect of the invention refers to an antibody or fragmentthereof produced, obtained or obtainable by the method of aspect F ofthe invention.

Aspect H: This aspect of the invention refers to the use of anAcinetobacter baumannii strain deficient in lipopolysaccharide (LPS)and/or an outer membrane vesicle (OMV) derived therefrom, for theproduction of antibodies or fragments thereof having affinity or bindingaffinity or capable of specifically binding such strain and/or OMV.

DETAILED DESCRIPTION OF THE INVENTION

The present invention refers to compositions and vaccines that consistof whole cells deficient in LPS and/or outer membrane vesicles ofAcinetobacter baumannii (A. baumannii) capable of conferring protectionagainst infection caused by infectious pathogens.

The authors of the present invention demonstrate that inactivated cellsof A. baumannii deficient in LPS and/or outer membrane vesicles from A.baumannii, upon being inoculated produce immunization, which providesprotection against posterior infection by said bacteria, whichdemonstrates the utility of these cells or strains as prophylacticvaccines against infections caused by A. baumannii.

A first aspect of the present invention refers to an Acinetobacter cellor strain that is deficient in LPS, hereinafter cell or strain of theinvention.

The species of Acinetobacter are strictly aerobic non-fermenting andnon-motile bacilli that are oxidase negative and appear in pairs bymicroscopy. They are distributed widely in nature, and are important insoil and contribute to its mineralization.

It is understood that “inactivated cell” in the present invention is acell that does not have the ability to replicate but that conserves itsimmunogenic capacity. The cells of the present invention are inactivatedprior to their inoculation to prevent their replication in the host, andtherefore prevent invention produced by their administration. Theinactivation of the cells of the invention can be performed usingdiverse methods known in the state of the art for example, although notlimited to, adsorption, heat, ultraviolet light, ionizing radiation,ultrasound, phenol, formol, formaldehyde, crystal violet,glyceraldehyde, ethylene oxide, propiolactone, ethylenamina,bromoethyleneamina or formalin. In a preferred embodiment, the cells ofthe invention are inactivating with formalin. In another preferredembodiment the cells of the invention are from the species Acinetobacterbaumannii and they are inactivated with formalin.

In a preferred embodiment of this aspect of the invention, thedeficiency in LPS can be achieved by partial or complete inactivation ofone or various cellular molecules of nucleic acids that encode theendogenous genes for the LPS subunits, particularly IpxA, IpxB and/orIpxC of LPS.

In another preferred embodiment of the invention, the cell or strain ofAcinetobacter deficient in LPS is obtained by deletions and/orinsertions of one or various nucleotides in nucleic acid sequencesencoding the gene involved in the biosynthesis of LPS and/or thesequences that control their expression. The deletions and/or insertionscan be generation by homologous recombination, insertion of transposons,or other adequate methods known in the state of the art.

In preferred embodiment of the invention, the sequence is inactivatede.g by construction of a suicide vector that contains the gene IpxA,IpxB, IpxC, IpxD, IpxK, IpxL and/or IpxM or any of their combination, orinterrupting with a marker gene for selection, transforming the targetcells with the vector and screening for positive cells that are negativefor LPS expression.

In another preferred embodiment of the invention, the cell or strain ofthe invention is preferably an A. baumannii cell, particularly anattenuated A. baumannii cell or other Acinetobacters; Acinetobacterbaylyi, A. beijerinckii, A. bereziniae, A. boissieri, A. bouvetii, A.brisouii, A. calcoaceticus, A. gerneri, A. guillouiae, A. grimontii, A.gyllenbergii, A. haemolyticus, A. indicus, A. johnsonii, A. junii, A.lwoffii, A. nectaris, A. nosocomialis, A. parvus, A. pittii, A.puyangensis, A. radioresistens, A. rudis, A. schindleri, A. soli, A.tandoii, A. tjernbergiae, A. towneri, A. ursingii or A. venetianus.

In this report, it is understood that Acinetobacter refers to thekingdom Bacteria, phylum Proteobacteria, class Gammaproteobacteria,order Pseudomonadales, family Moraxellaceae.

It is understood that “cells or strains of the Acinetobacter baumannii”in the present invention are those cells pertaining to the domainBacteria, phylum Proteobacteria, class Gammaproteobacteria, orderPseudomonadales, family Moraxellaceae, genus Acinetobacter, speciesAcinetobacter baumannii.

It is understood that “lipopolysaccharide (LPS) or lipooligosaccharide”is a component that is found on the external membrane of variousGram-negative bacteria. The term LPS is used often and interchangeablywith “endotoxin”, due to its history of discovery. LPS consists of apolysaccharide chain and the rest is lipid, known as lipid A, which isresponsible for the endotoxin activity. The polysaccharide chain isvariable between different bacterias and determines the serotype.Endotoxin is of approximately 10 kDa in size, but can form largeaggregates of up to 1000 kDa. Humans are able to produce antibodiesagainst LPS, but in general these antibodies can only protect againstbacteria of a specific serotype. Endotoxin is responsible for many ofthe clinical manifestations of infections caused by Gram-negativebacteria such as Neisseria meningitidis and Acinetobacter baumannii.

Composition of the Invention

A second aspect of the invention refers to a composition, hereinaftercomposition of the invention comprising:

-   -   a) a cell or strain of the invention, and    -   b) optionally a sequence of nucleotides and/or amino acids or        polypeptides.

In a more preferred embodiment of this aspect of the invention, thenucleic acid and/or the amino acid sequence or polypeptide isrecombinant.

In a still more preferred embodiment, the polypeptide is selected from

-   -   I) the peptide sequence SEQ ID NO: 27 (putative ferric        siderophore receptor (A. baumannii ATCC 17978; accession number        YP_001084684)) or a fragment thereof, wherein the fragments are        biologically active fragments, preferably selected from the list        consisting of SEQ ID NO: 1 to SEQ ID NO: 11, or any of its        combinations, or sequences having at least 85% sequence identity        with the peptide sequences SEQ ID NO: 1 to SEQ ID NO: 11, and/or    -   II) the peptide sequence SEQ ID No 28 (putative ferric        hydroximate siderophore receptor (A. baumannii ATCC 17978;        accesión number YP_001084696)) or a fragment thereof, wherein        the fragments are biologically active fragments, preferably        selected from the list consisting of SEQ ID NO: 12 to SEQ ID NO:        23, or any of its combinations, or sequences having at least 85%        sequence identity with the peptide sequences SEQ ID NO: 12 to        SEQ ID NO: 23.

In a still more preferred embodiment, the composition of the inventioncomprises the amino acid sequence SEQ ID NO: 28 and the amino acidsequence SEQ ID NO: 27.

In another preferred embodiment, the composition of the inventioncomprises a fusion protein that consists of at least 2, preferably 3,more preferably 4, amino acid sequences from the following listconsisting of: SEQ ID NO: 1 to SEQ ID NO: 23 or a variant of thesesequences having at least 85% identity with the sequences SEQ ID NO: 1to SEQ ID NO: 23.

In another preferred embodiment, the fusion protein comprises the aminoacid sequence SEQ ID NO: 24 or the amino acid sequence SEQ ID NO: 25.

In another preferred embodiment, the composition of the inventioncomprises a nucleotide sequence, hereinafter nucleotide sequence of theinvention, capable of transcribing an amino acid sequence described inthe invention. More preferably, the nucleotide sequence is SEQ ID NO:26.

The fragments described previously differ in amino acid sequence by atleast one amino acid. The most preferred variations are those having atleast 85%, or more, including 90%, 93% or more, and preferably 95% ormore, 96% or more, 97% or more, 98% or more, 99% or more of sequenceidentity with any of the polypeptides shown in SEQ ID NO:1 to SEQ IDNO:25. More preferably, the invention refers to a sequence variantcharacterize by at least one (at least two, at least three, at leastfour) mutation(s) in relation to any of the polypeptides SEO ID NO:1 toSEC ID NO:25. In agreement with the invention as described in thedescriptive section, mutation can refer to any mutation selected byinsertion(s), deletion(s), and substitution(s). Preferablysubstitution(s).

In another preferred embodiment, the composition of the inventioncomprises an expression vector (hereinafter expression vector of theinvention), comprising a nucleotide sequence of the invention.

In another preferred embodiment, the composition of the invention alsocomprises outer membrane vesicles, hereinafter outer membrane vesiclesof the invention, deficient in LPS.

In another preferred embodiment, the composition of the invention alsocomprises at least one or the proteins purified from the membrane of A.baumanni with amino acid sequence SEQ ID NO: 29, SEQ ID NO: 30, SEQ IDNO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40,SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO:45, SEQ ID NO: 46; SEQ ID NO: 47; SEQ ID NO: 48; SEQ ID NO: 49; SEQ IDNO: 50; SEQ ID NO: 51; SEQ ID NO: 52; SEQ ID NO: 53; SEQ ID NO: 54; SEQID NO: 55; SEQ ID NO: 56; SEQ ID NO: 57; SEQ ID NO: 58; SEQ ID NO: 59;SEQ ID NO: 60; SEQ ID NO: 61; SEQ ID NO: 62; SEQ ID NO: 63; SEQ ID NO:64; SEQ ID NO: 65; SEQ ID NO: 66; SEQ ID NO: 67; SEQ ID NO: 68; SEQ IDNO: 69; SEQ ID NO: 70; SEQ ID NO: 71; SEQ ID NO: 72; SEQ ID NO: 73; SEQID NO: 74; SEQ ID NO: 75; SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78,SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO:83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ IDNO: 88, y SEQ ID NO: 89, or any of their combinations.

More preferably, at least one of the proteins of the outer membrane ofA. baumannii is obtained by a process comprising:

-   -   a. Inoculuting one liter of Mueller-Hinton broth with a colony        of A. baumannii ATCC 19606;    -   b. incubating the culture until an optical density of 0.6 at 600        nm;    -   c. Washing bacterial cells with 30 ml of 10 mM phosphate buffer        pH, 7.2;    -   d. Centrifuge at 6000×g for 10 min;    -   e. Resuspend the bacterial pellet sin 10 ml of 10 mM phosphate        buffer pH 7.2 and lysing by sonication 5 times for 1 minute;    -   f. Eliminate unlysed cells by centrifugation at 6000×g for 5        minutes;    -   g. Centrifuge the supernatant at 4° C. at 20000×g for one hour;    -   h. Eliminate proteins of the external membrane by solubilising        with 5 ml of 2% N-lauraylsarcosinante in 10 mM phosphate buffer        pH 7.2 for 30 minutes at 37° C.;    -   i. Precipitate the insolube fraction (that contains the outer        membrane proteins) by centrifuging at 4° C. at 20000×g for one        hour;    -   j. Wash the pellet once with 2 ml 62.5 mM Tric-HCl ph, 6.8 and        centrifuge at 4° C. for one hour;    -   k. Resuspend the pellet in a solution of 5% SDS and precipitate        with methanol/chloroform; and    -   l. Resuspend the pellet in sterile PBS.

In another preferred embodiment of the invention, the composition of theinvention is a pharmaceutical composition, more preferably alsocomprises an acceptable pharmaceutical vehicle, and still morepreferably, also comprises another active ingredient.

In another preferred embodiment, the composition of the invention alsocomprises an adjuvant. In another preferred embodiment, the compositionof the invention is a vaccine.

In the context of the present invention, the term “vaccine” refers to anantigenic preparation employed for inducing an immune response to adisease. They are prepared from antigens that, once inside the host,provoke an immune response through the production of antibodies, andgenerate immunologic memory producing transient or permanent immunity.It is noted that as used herein the term “vaccine” can also beunderstood as a preparation from antibodies or fragments thereofsuitable for the therapeutic treatment or for passive immunization of aninfection caused by an Acinetobacter strains, in particular from an A.baumannii strain.

Nucleotide Sequence and Expression Vector

A third aspect of the invention refers to a nucleotide sequence,hereinafter second nucleotide sequence of the invention, which encodesany of SEQ ID NO 1 to 25, 27 or 28 or any combination thereof. Saidsecond nucleotide sequence of the invention also includes nucleic acidsequence SEQ ID NO: 26.

A fourth aspect of the invention refers to an expression vector,hereinafter expression vector of the invention, comprising the secondnucleotide sequence of the invention.

In a preferred embodiment of this aspect, the cell of the inventioncomprises the expression vector of the invention.

The nucleic acid can be localized in a recombinant vector. Preferably,the recombinant vector is a prokaryotic vector, that is a vector thatcontains elements for replication and/or integration into the genome ofprokaryotic cells. Preferably, the recombinant vector contains thenucleic acid molecule of the present invention operatively linked to anexpression control sequence. The control sequence is preferably asequence controlling the active expression in Acinetobacter,particularly in A. baumannii. The vector can be an extrachromosomicvector adequate for integration in the chromosome. Examples of such asvectors are known by experts in the field, for example in Sambrook etal. supra.

Outer Membrane Vesicles of the Invention

A fifth aspect of the invention refers to an outer membrane vesicle thatis deficient in LPS. In a preferred embodiment of this aspect, the outermembrane vesicle is obtained from the cell or strain of the invention.

Use of the Cells, Vesicles and the Compositions of the Invention

A sixth aspect refers to the composition of the invention for use as amedicament, or alternatively, to the use of the composition of theinvention for the manufacture of a medicament.

The term “medicament” or “pharmaceutical composition” as used in thisreport, makes reference to any substance used for the prevention,alleviation, treatment or cure of a disease in man or animals. Thecontext of the present invention refers to a composition comprising thecomposition of the invention. This composition of the inventioncomprises inactivated cells of the genus Acinetobacter and/or outermembrane vesicles of the same in a quantity therapeutically effective,that is able to inducer an immune response in the organism in which theyare administered against an organism of the genus Acinetobacter. Theterm “medicament” or “pharmaceutical composition” therefore is known asa vaccine.

The dosing for obtaining the effective therapeutic quantity depends on avariety of factors such as for example, age, weight, sex, tolerante, . .. of the mammal. As used in this description, the “effective therapeuticquantity” refers to the quantity of inactive cells of the genusAcinetobacter and/or outer membrane vesicles that produce the desiredeffect, and in general are determined by the therapeutic effect that isdesired.

A seventh aspect of the invention refers to the composition of theinvention for the prevention, the improvement or the treatment of aninfection caused by A. baumannii, en a mammal, or alternatively, to theuse of the composition of the invention for the elaboration of amedicament for the prevention, improvement or treatment of an infectioncaused by A. baumannii in a mammal.

It is understood by “disease produced by organisms of the genusAcientobacter” those diseases in which the causal agent of the pathologyis from the genus Acinetobacter, or any of its metabolic products. Thegenus Acinetobacter produces diverse pathologies for example but notlimited to, bacteremia, meningitis, urinary tract infections, skin andsoft tissue infections, surgical site infections and pneumonia. Forthese reasons, one of the more preferred forms, diseases produced byorganisms of the genus Acinetobacter are selected from a list thatconsists of bacteremia, meningitis, urinary tract infections, skin andsoft tissue infections, surgical site infections and pneumonia.

The medicaments and compositions of the invention can be used alone orin combination with other medicaments or compositions for the treatmentof diseases produced by organisms from the genus Acinetobacter.

Both the medicaments and the compositions of the invention can alsoinclude pharmaceutically acceptable vehicles or excipients

The medicaments and compositions of the invention may be used eitheralone or in combination with other medicaments or compositions for thetreatment or prevention of diseases caused by organisms of the genusAcinetobacter.

The term “excipient” makes reference to a substance that helps in theabsorption of the elements of the composition of the medicaments of theinvention, stabilizing said elements, activating or helping thepreparation of the medicament such that it provides consistency orflavours that make it more palatable. The excipients can maintain theingredients together, like for example is the case with starches,sugars, cellulose, sweetners, coloring agents, the function ofprotecting the medicament, for example isolating it form air and/orhumidity, the function of filling the pill, capsule or any other form ofpresentation, for example, the case of dibasic calcium phosphate, thefunction for facilitating dissolution of the components and theirabsorption in the intestine, without excluding other types of excipientsdescribed in this paragraph.

The vehicle, in the same way as the excipient, is a substance that isused in the medicament to dilute any of the components of the presentinvention to a desired volume or weight. The pharmaceutically acceptablevehicle is an inert substance or of similar action to any of theelements of the present invention. The function of the vehicle is tofacilitate the incorporation of other elements, permit better dosing andadministration and give consistency and form to the medicament. When theform of presentation is liquid, the pharmaceutically acceptable vehicleis the diluent.

The adjuvants and pharmaceutically acceptable vehicle that can be usedin the composition of the invention are those vehicles known by expertsin the field.

In this report, the term “adjuvant” refers to any agent that does notposes antigenic activity in and of itself, that can be used to stimulatethe immune system to increase the response to a vaccine. There are manyadjuvants, for example but not limited to, aluminium phosphate,aluminium hydroxide, toll-like receptor agonists, cytokines, squaline,Freunds incomplete and complete adjuvants. In a preferred form of thisaspect of the invention, the adjuvant is selected for a list thatconsists of aluminium phosphate, aluminium hydroxide, toll-like receptoragonists, cytokines, squaline, Freunds incomplete and completeadjuvants. In a still more preferred form of this aspect of theinvention, the adjuvant is aluminium phosphate.

As used here, the term “active ingredient”, “active substance” or“pharmaceutically active substance” or “pharmaceutically activeingredient” refers to any component that potentially providespharmacological activity or other different effect in the diagnosis,cure, alleviation, treatment or prevention of a disease or that affectsthe structure or function of the human or animal body. The term includesthose components that promote a chemical change in the elaboration ofthe drug and are present in the same and a modified form that providesspecific activity or the effect.

An eighth aspect of the invention refers to the composition of theinvention for conferring protection against an infection caused by A.baumannii en a mammal, or alternatively, the use of the composition ofthe invention in the elaboration of a medicament for conferringprotection against an infection caused by A. baumannii in a mammal.

Another aspect of the invention refers to the fusion protein or peptideof the invention or the composition of the first or second aspect of theinvention or the pharmaceutical composition of the invention that can beadministered once or various, such as two, three, four, five, six,seven, eight, nine or ten or more times. There are no particularlimitations relative to the quantity of the active ingredient per dose.

An additional aspect of the present invention refers to a compositionthat consists of an antibody or fragment thereof that is capable ofbinding to SEQ ID NO: 27 or SEQ ID NO: 28 or a fusion protein as definedin the second aspect or the fusion protein of the invention, whereinpreferably said composition is a pharmaceutical composition, preferablya vaccine, and wherein said pharmaceutical composition is used in thetreatment or prevention of an infection caused by A. baumannii.

A ninth aspect of the invention refers to an antibody or an activefragment thereof obtainable by immunization of a mammal with thecomposition of the first or second aspect of the invention or with thefusion protein of the invention, preferably said antibody or activefragment consists of a composition in which preferably said compositionis a pharmaceutical composition and said pharmaceutical composition isused as a therapy, particularly for the treatment of infections causedby A. baumannii.

Method for Preparing an Acinetobacter Cell Following the Invention

A tenth aspect of the invention refers to a method for preparing a cellof A. baumannii as described above.

Acinetobacter

In agreement with this aspect, this method consists of the steps to (i)provide a bacterial cell deficient in LPS, particularly a cell ofAcinetobacter, (ii) insert a recombinant nucleic acid molecule in saidbacterial cell, encoding said nucleic acid molecule a fusion peptidethat consists of (a) at least on domain of the polypeptide wherein saiddomain is capable of producing an immune response in mammal and (b) adomain of escape for the phagolysosome, and (iii) culture the obtainedcell in agreement with step (ii) in adequate conditions. Preferably, acell capable of expressing said nucleic acid is obtained. Morepreferably, the cell is a cell of A. baumannii.

In agreement with the additional aspect, this method consists of step of(i) providing a bacterial cell deficient in LPS, particularly anAcinetobacter cell, (ii) inserting a recombinant nucleic acid moleculein said bacteria, encoding said nucleic acid molecule a peptide orpolypeptide for escape from the phagolysosome, and (iii) culture theobtained cell in agreement with step (ii) in adequate conditions.

If desired, the method of the present invention consists of inserting atleast one recombinant nucleic acid molecule in the bacterial cell, saidmolecule encoding a peptide or polypeptide capable of producing animmune response in mammals.

It is understood that “infection” in the present invention is thatpathology generated by the invasion or colonization of any host tissueby any organisms of the genus Acinetobacter, preferably Acinetobacterbaumannii.

It is understood that “soft tissue” in the present invention, is allnon-bony tissue of an organism.

The term “prevention” as understood in the present invention consists ofavoiding the appearance of damage whose cause is cells of the genusAcinetobacter, or any derivative or metabolic product of the same.

The term “antigen” in the report refers to a molecule (generally aprotein or polysaccharide) that can induce the formation of antibodies.There many different types of molecules that can act as antigens, suchas proteins, peptides, polysaccharides, and more rarely other moleculessuch as nucleic acids.

The term “resistance” refers to any mechanism of defense developed bybacteria against a drug. The mechanisms of resistance acquired andtransmitted by bacteria are the most important and consist primarily of:the production of enzymes that inactivate antibiotics, appearance ofmodifications that impede the arrival of the drug to its target and/oralteration of the target itself. One bacterial strain can developvarious resistance mechanisms against one or many antibiotics and in thesame way an antibiotic can be inactivated by distinct mechanisms fromdiverse bacterial species.

The terms “polynucleotide” and “nucleic acid” are used interchangeably,referring to polymeric forms of nucleotides of any length, both of DNAand RNA

The terms “amino acid sequence”, “peptide”, “oligopeptide”,“polypeptide” and “protein” are used interchangeably, and refer to apolymeric form of amino acids of any length that can be chemically orbiochemically modified.

During the description of the claims, the word “comprising” and itsvariants does not intend to exclude other technical characteristics,additives, components or steps. For those expert in the state of theart, other objects, advantages and characteristics of the invention arereleased in the section of the description and practice of theinvention. The following examples and drawings are provided asillustrations, and are not intended to limit the present invention.

DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B. Stability and endotoxin content of IB010. (FIG. 1A)Genomic DNA from three independent cultures of ATCC 19606 and IB010 wasextracted and amplified using primers specific for the IpxD gene. Theband corresponding to approximately 1000 Kb corresponds to the intactIpxD gene, whereas the faster migrating band corresponds to the IpxDgene with a deletion of 462 nucleotides. (FIG. 1B) Endotoxin levels ofATCC 19606 and IB010 determined by the Limulus Amebocyte Assay. Barsrepresent the median values of three independent cultures, and errorbars represent the standard error of the mean. EU; endotoxin units.

FIGS. 2A-2D. Antibody response to immunization with IB010. Serum sampleswere collected from ATCC 19606 vaccinated, IB010 vaccinated and controlmice before vaccination (Day 0) and at day 7 and 21 after the firstimmunization, and levels of antigen specific total IgG (FIG. 2A) and IgM(FIG. 2B) were measured by ELISA (n=8 mice/group). IgG1 (FIG. 2C) andIgG2c (FIG. 2D) levels were measured in 21-day serum were measured byELISA in ATCC 19606 vaccinated, IB010 vaccinated and control mice. Inall panels box and whisker plots represent the interquartile ranges andranges, respectively, and horizontal lines represent median values.*p<0.05 compared to levels in control mice at the same time point,#p<0.05 compared to 7-day samples from the same experimental group, †p<0.05 compared to 21-day samples in ATCC 19606 vaccinated mice.

FIG. 3. Effect of vaccination on tissue bacterial loads. Immunized andcontrol mice were infected with 2.0×10⁶ cfu (300×LD₅₀) of the ATCC 19606strain and spleen bacterial loads were determined 12 hourspost-infection (n=8 mice/group). Data points represent bacterial loadsfrom individual mice, and horizontal lines represent median values fromgroups of mice. *p<0.05 compared to control mice.

FIG. 4. Effect of vaccination on post-infection pro-inflammatorycytokine levels. Immunized and control mice were infected with 2.0×10⁶cfu (300×LD₅₀) of the ATCC 19606 strain and serum levels of IL-1β,TNF-α, and IL-6 were determined (n=8 mice/group). Data points representcytokine levels from individual mice, and horizontal lines representmedian values from groups of mice. *p<0.05 compared to control mice,#p<0.05 compared to ATCC 19606 vaccinated mice.

FIGS. 5A and 5B. Effect of vaccination on survival in a mouse model ofdisseminated A. baumannii infection. Vaccinated and control mice wereinfected with 2.25×10⁶ cfu (340.9×LD₅₀) of the ATCC 19606 strain (FIG.5A) or 1.05×10⁶ cfu (2.18×LD₅₀) of the A. baumannii clinical isolateAb-154 FIG. 5B), and survival was monitored over the following 7 days(n=8 mice/group). *p<0.05 compared to control mice.

FIG. 6. Protein profile of the A. baumannii OMVs without LPS. Thestrains ATCC 19606 and IB010 for the production of OMVs after culturing24, 48 and 72 hours. After the purification of OMVs the amount ofprotein was quantified using Bradford method and 10 mcg of the proteinwas visualized in a 10% polyacrylamide gel with Coomassie stain.

FIG. 7. Effect of 2,2 Bipyridyl, iron chelator, in the protein profileof OMVs. The strains ATCC 19606 and IB010 were used to analyze theeffect of 2,2 bypiridyl (BIP) on the protein profile of the OMVs afterculturing for 24 hours. In the case of the ATCC 19606 strain aconcentration of 200 mcM was used while in the case of IB010 100 and 150mcM was used. After the purification of OMVs the amount of protein wasquantified using Bradford method and 10 mcg of the protein wasvisualized in a 10% polyacrylamide gel with Coomassie stain.

FIG. 8. Effect of 2,2 Bipyridyl, iron chelator, on the production ofOMVs. The strains ATCC 19606 and IB010 were used to analyze the effectof 2,2 bypiridyl (BIP) on the production of the OMVs after culturing for24 hours. In the figure the total protein content of the OMVs is shownafter treatment with Bip and measuring the concentration with Bradford.

FIG. 9. Visualization of OMVs. OMVs purified form ATCC 19606 were fixedusing glutaraldehyde at 1.6% and stained with osmium tetroxide and leadand uranium and visualized by electron microscopy.

FIG. 10. Visualization of purified OMVs. OMVs purified form IB010 werefixed using glutaraldehyde at 1.6% and stained with osmium tetroxide andlead and uranium and visualized by electron microscopy.

FIG. 11. Protein profile of OMVs without LPS. The strains IB010 and 167Rwere used to produce OMVs and the amount of protein was quantified fromeach sample. Ten mcg of the protein was visualized in a 10%polyacrylamide gel with Coomassie stain.

EXAMPLES OF THE INVENTION Example 1

Ethics Statement

All experiments involving the use of animals were approved by theUniversity Hospital Virgen del Rocío Committee on Ethics andExperimentation (Evaluation code: 2013PI/296). In all experiments,efforts were made to minimize suffering, and any animals appearingmoribund during the course of experimentation were immediatelyeuthanized using thiopental.

Bacterial strains. A. baumannii ATCC 19606 is an antibiotic susceptiblereference strain. An LPS-deficient derivative of ATCC 19606 was obtainedby plating an overnight culture of ATCC 19606 on Mueller Hinton agarcontaining 10 mg/I of colistin, as described previously (ClinicalLaboratory Standards Institute 2013) Strains with mutations in the genesinvolved in LPS biosynthesis were identified by sequencing the IpxA,IpxC and IpxD genes of the colistin resistant mutants that were presentafter overnight growth at 37° C. A strain with a large deletion in theIpxD gene was identified and designated IB010. Resistance to colistinwas confirmed by broth microdilution according to Clinical LaboratoryStandard Institute guidelines [23]. Absence of LPS was confirmed bymeasuring the endotoxin levels of three independent cultures of eachstrain using the QCL-1000 Limulus Amebocyte Assay (Lonza) according tothe manufacturer's instructions. The Ab-154 strain is a previouslycharacterized A. baumannii clinical isolate (Gautom, 1997. J. Clin.Microbiol. 35, 2977-2980).

Vaccine preparation and mouse immunization. The IWC vaccines (bothLPS-containing and LPS-deficient) were prepared as described based on apreviously described method (Moffatt et al., 2010. Antimicrob. AgentsChemother. 54, 4971-4977). Briefly, the ATCC 19606 and IB010 strainswere grown in Mueller-Hinton broth to OD₆₀₀ of 0.8. In the case ofIB010, 10 μg/ml of colistin were added to the culture. In order toconfirm that no reversion to wild type occurred during growth of IB010,three independent cultures of ATCC 19606 and IB010 were grown, andgenomic DNA was isolated from each culture using the QIAmp DNA Mini Kit(Qiagen). The IpxD specific primers 5′ GCTAATTGGTGAAGGTAGTC 3′ and 5′GACGAATCGTTTGAATCTGC 3′ were used to amplify genomic DNA from thecultures in order to confirm that the deletion in IpxD of IB010 waspresent after growth.

For vaccine preparation, bacteria were washed extensively in phosphatebuffer saline before inactivation in 0.5 M formalin for 18 h withshaking at room temperature. Complete inactivation of the bacteria wasconfirmed by plating on blood agar. The concentration of inactivatedcells was adjusted to 1×10¹⁰ cells/ml and combined 1:1 (v/v) with thealuminium-based adjuvant, Alhydrogel 2% (w/v) (InvivoGen). Vaccinationwas carried out in 6 to 8-week-old, female C57BL/6 mice by intramuscularinjection of 100 μl of the vaccine into each quadriceps muscle on days 0and 14. Control mice were injected similarly with a mixture of phosphatebuffer saline and adjuvant.

Mouse model of A. baumannii infection. A mouse model of sepsispreviously developed by our group and used for the evaluation ofvaccines against A. baumannii was used to characterize the efficacy ofthe (Batson et al., 1950. J. Exp. Med. 91, 219-229; Rodriguez-Hernandezet al., 2000. J. Antimicrob. Chemother. 45, 493-501). This modelproduces a disseminated infection after intraperitoneal instillation ofthe inoculum, typically resulting in death within 24 to 48 hours. Forpreparation of the inocula, A. baumannii strains were grown for 18 h at37° C. in Mueller-Hinton broth cultures and adjusted to the appropriatedconcentration in physiological saline as described previously (Moffattet al., 2010. Antimicrob. Agents Chemother. 54, 4971-4977; Martin etal., 1998. J. Immunol. Methods 212, 187-192). Bacterial concentrationsof the inocula were determined by plating on blood agar. Mice wereinfected on day 21 (one week after the second immunization) byintraperitoneal injection with 0.5 ml of the bacterial suspension andsurvival was monitored for 7 days.

Spleen bacterial loads and serum cytokine levels. Post-infectionbacterial loads were determined in vaccinated and control mice 12 hafter infection. Mice were euthanized with an overdose of thiopental andafter collection of blood samples from the retro-orbital sinus, spleenswere aseptically removed, weighed and homogenized in 2 ml ofphysiological saline. Serial log dilutions were plated on blood agarplates for bacterial quantification. Serum levels of interleukin-1β(IL-1β), tumor necrosis factor alpha (TNF-α), and interleukin-6 (IL-6)were determined in mice at 12 h post-infection using BD OptEIA mousekits (BD Biosciences).

Enzyme-linked immunosorbent assays (ELISAs). For indirect enzyme-linkedimmunosorbent assays (ELISAs), 96-well plates were coated with 5×10⁷bacterial cells/well in phosphate buffer saline by incubating at 4° C.overnight. ELISAs were performed using sera collected on days 0, 7 and21 as described previously [28]. Antibody titers were measured againstthe strain which was used to immunize the mouse, and were defined as thedilution in which spectrophotometric readings were at least 0.1 unitsabove background wells (wells containing no serum).

Statistical analysis. Antibody titers, bacterial loads, and cytokinelevels were compared using the Kruskal-Wallis H test and theMann-Whitney U test for independent samples, and the Friedmann andWilcoxon tests for dependent samples. The Bonferroni correction wasapplied when appropriate. Survival data were compared using the log-ranktest. All statistics were performed using SPSS version 15.0 software(SPSS Inc.), and a p value of 0.05 was considered significant.

Results

Selection of an LPS-deficient strain for vaccine development. Growth ofATCC 19606 in the presence of 10 μg/ml colistin resulted in numerouscolistin-resistant derivatives with mutations in the IpxA, IpxC and IpxDgenes (data not shown). One of these strains, IB010, contained a largedeletion of 462 nucleotides in the IpxD (nucleotides 104-565) gene andwas chosen for further use in vaccine studies. We reasoned that on thebasis that the strain contained a large deletion, this strain would beless likely to revert to wild type during growth than strains containingsingle nucleotide changes or small deletions in the LPS biosynthesisgenes. Broth microdilution experiments demonstrated that the minimuminhibitory concentration of the ATCC 19606 strain was 0.25 μg/mland >128 μg/ml for IB010, demonstrating that, similar to resultsdescribed previously, mutations in IpxD can result in resistance tocolistin (Moffatt et al., 2010. Antimicrob. Agents Chemother. 54,4971-4977). In order to ensure that the IB010 was genetically stableduring growth, genomic DNA from three independent cultures of ATCC 19606and IB010 were amplified with IpxD-specific primers to confirm that thedeletion was present. As shown in FIG. 1A, a band corresponding to themutated IpxD gene of IB010 containing a deletion of 462 nucleotides waspresent after amplification from all IB010 cultures indicating that noreversion had occurred. Phenotypic loss of LPS and reduction inendotoxin levels were characterized by the Limulus Amebocyte Assay forATCC 19606 and IB010, and demonstrated that mutation in the IpxD generesulted in a dramatic reduction in endotoxin levels to >1 EU per 10⁶cells (FIG. 1B).

Antibody response to the LPS-deficient IWC vaccine. Formalin treatmentof ATCC 19606 and IB010 resulted in no viable bacteria, indicatingcomplete bacterial inactivation. In order to quantify the antibodyresponse produced by immunization with inactivated IB010, indirectELISAs were performed using sera collected from negative control mice(immunized with PBS and adjuvant) and mice vaccinated with 1×10⁹inactivated IB010 cells. As a positive control, one group of mice wasimmunized with 1×10⁹ inactivated ATCC 19606 cells on the basis that wehave previously shown that immunization with these cells induces arobust immune response and produces protective immunity againstexperimental infection (McConnell y Pachon, 2010. Vaccine 29, 1-5). Asshown in FIG. 2A, immunization with inactivated IB010 eliciteddetectable levels of antigen-specific total IgG in all mice seven daysafter a single intramuscular administration, and these antibody levelswere significantly increased upon boosting with a second administrationof the vaccine (p=0.03 Wilcoxon test). Total IgG titers in micereceiving two administrations of inactivated IB010 vaccine were similarto titers in mice receiving the vaccine containing inactivated wild typecells (p=0.726 Mann Whitney U test). Control mice had no detectableantigen-specific IgG at any point. In contrast, IgM levels were similarbetween mice immunized with the inactivated IB010 vaccine and micereceiving inactivated wild type cells seven days after a singleadministration (p=0.186 Mann Whitney U test), however seven days after asecond immunization there was no detectable antigen-specific IgM inIB010-vaccinated mice whereas all mice immunized with inactivated wildtype cells had detectable levels of IgM (FIG. 2B).

Levels of the IgG subtypes IgG1 and IgG2c, the IgG2a homolog in C57BL/6(Martin et al., 1998. J. Immunol. Methods 212: 187-192), were determinedin 21-day serum (FIGS. 2C and D). Both groups of mice receiving theinactivated vaccines had significant levels of IgG1 and IgG2c comparedto control mice (p<0.001; Mann-Whitney U test). Interestingly, IgG1titers were significantly higher in IB010-vaccinated mice compared toATCC 19606-vaccinated mice (p=0.003; Mann-Whitney U test), whereas IgG2ctiters were similar between these groups. These results indicate thatboth Th1 and Th2 responses are elicited by the inactivated IB010 vaccinesimilar to what was previously shown for the inactivated ATCC 19606vaccine (McConnell y Pachón, 2010. Vaccine 29, 1-5).

Effect of vaccination on post-infection bacterial loads. In order tocharacterize the effect of vaccination on post-infection tissuebacterial loads, we employed a mouse model previously developed by ourgroup for the characterization of vaccine for preventing infection by A.baumannii (McConnell et al., 2011. Infect. Immun. 79, 518-526; McConnellet al., 2011. Vaccine 29: 5705-5710; McConnell y Pachon, 2010. Vaccine29, 1-5). This model rapidly produces a disseminated infection in whichbacteria are detected in distal organs as soon as one hourpost-infection [16]. Vaccinated and control mice were infected with2.0×10⁶ cfu (300×LD₅₀) of the ATCC 19606 strain, and 12 hours afterinfection spleen bacterial loads were determined (FIG. 3). IB010vaccination reduced the number of bacteria in spleens approximately1000-fold compared to control mice (p<0.05; Mann-Whitney U test). Spleenbacterial loads in IB010 vaccinated mice were not significantlydifferent than in mice immunized with inactivated ATCC 19606 cells.

Effect of vaccination on post-infection serum cytokine levels andsurvival. In order to characterize the effect of immunization with theinactivated LPS deficient vaccine on cytokine levels, sera werecollected from vaccinated and control mice 12 h post-infection and thelevels of IL-1β, IL-6 and TNF-α were determined (FIG. 4). Levels of allthree cytokines were significantly lower in both groups of vaccinatedmice than in control mice (p=0.003 for IL-1β, IL-6 and TNF-α;Mann-Whitney U test), suggesting that vaccinated mice did not experiencethe pro-inflammatory cytokine release associated with the development ofseptic shock.

Vaccine efficacy was tested by infecting immunized and control mice with2.25×10⁶ cfu (340.9×LD₅₀) of the ATCC 19606 strain seven days after thesecond immunization, and survival was monitored over seven days (FIG.5). All mice vaccinated with the IB010 vaccine were protected fromchallenge, whereas all control mice died within 48 hours (P<0.001;log-rank test). As expected, all mice immunized with the ATCC 19606strain survived challenge, similar to results that were previouslyreported (McConnell et al., 2011. Infect. Immun. 79, 518-526; McConnellet al., 2011. Vaccine 29: 5705-5710; McConnell y Pachón, 2010. Vaccine29, 1-5). In order to determine if vaccination with IB010 could protectagainst heterologous challenge with an unrelated strain, immunized andcontrol mice were infected with 1.05×10⁶ cfu (2.18×LD₅₀) of thepreviously characterized A. baumannii clinical isolate Ab-154 [29]. Onceagain, all immunized mice survived challenge whereas control micesuccumbed to infection within 48 hours (p<0.001; log-rank test),indicating that immunization with IB010 can provide cross protectionagainst challenge with a heterologous strain.

In conclusion, these results provide important information regarding thedevelopment of a vaccine for the prevention of infections caused by A.baumannii based on whole bacterial cells lacking LPS. These results mayalso provide insights into the possibility of developing vaccines forother bacterial species based on strains lacking LPS.

Example 2

This example relates to the development of a vaccine against A.baumannii based on OMVS purified from said cultures of mutants withoutLPS.

To carry out this objective, the strains ATCC 19606T and its mutantwithout LPS IB010, which was generated in our laboratory from the ATCC19606T strain and contains a deletion of 462 nucleotides betweenpositions 103 and 565 of the gene IpxD.

Upon realizing the purification of the OMVs of said strains thefollowing protocol was used:

-   -   Strains are refreshed on blood agar or MHBII plates with        colistin at 10 mcg/ml and grown overnight at 37° C.    -   A liquid culture is used to growth ATCC 19606 or IB010. They are        cultured with aeration at 180 rpm at 37° C. overnight.    -   The next day cultures of 50 or 100 ml or 1 L in MHB are made and        incubated overnight at 37° C. with aeration (180 rpm)    -   After incubation, the cells are centrifuged at 4000 rpm during        30 min at 4° C.    -   Next, the supernatant is filtered with a 0.22 micron filter.    -   Afterwards, the OMVs are precipitated by ultracentrifugation for        90 minutes at 30000 rpm at 4° C.

Finally, the pellet is resuspended in PBS and the absence of viablebacteria is confirmed by plating. The OMVs are stored at −80° C.

(Protocol adapted from McConnell M J et al. 2011 Aug. 5; 29(34):5705-10)

Using the previous protocol different purifications of OMVs have beenperformed.

-   -   Purifications of OMVs of the strains ATCC 19606 and IB010 for        the production of OMVs after culturing 24, 48 and 72 hours.        After the purification of OMVs the amount of protein was        quantified using Bradford method and 10 mcg of the protein was        visualized in a 10% polyacrylamide gel with Coomassie stain.    -   In addition, purification have been performed with OMVs from the        strain ATCC 19606 and IB010 in the presence and absence of Bip,        which is an iron chelator, with the objective of verifying if        the presence of the chelator resulted in the increased        expression of proteins related with iron metabolism, for        example, siderophore receptors. In this case, OMVs were purified        for the quantification of proteins and for Coomassie staining of        acrylamide gels and for the visualization of OMVs by electron        microscopy.

Finally, OMVs were purified form the LPS mutant of a clinical isolate ofA. baumannii Ab-167 which contains an ISAba1 insertion in the IpxC gene.Proteins were quantified and visualized on acrylamide gels by Coomassiestaining. And proteins were quantified by Bradford and 2D Quant kit.

Example 3. Purification of Outer Membrane Proteins

A. baumannii ATCC 19606 was grown in 1 liter of Mueller-Hinton broth toan optical density at 600 nm (OD600) of 0.6, and pelleted bacteria wereresuspended in 10 ml of 10 mM phosphate buffer, pH 7.2, and lysed bysonication. Unlysed cells were removed by centrifugation at 4,000×g for5 min, and the supernatant was centrifuged at 20,000_g for 1 h to pelletcell envelopes. Inner membranes were selectively solubilized with 5 mlof 2% N-laurylsarcosinate by incubation at 37° C. for 30 min. Theinsoluble fraction was pelleted by centrifugation at 20,000_g for 1 hand then washed with 2 ml of 62.5 mM Tris-CI, pH 6.8.

Endotoxin was extracted from the preparation by use of a cold detergentwash step in which proteins were resuspended in 5% SDS and incubated at4° C. for 10 min. SDS and endotoxin were subsequently removed byprecipitating in methanol chloroform and resuspended in PBS.

Addition of the Adjuvant

The purified proteins at a concentration of 500 mcg/ml were mixed withaluminum phosphate adjuvant at a 1:1 ration.

CLAUSES

1. An Acinetobacter cell deficient in LPS.2. The Acinetobacter cell according to the preceding claim, obtainedthrough partial or complete inactivation of one or various of thenucleic acids encoding the endogenous LPS biosynthesis genes.3. The Acinetobacter cell according to the preceding claim, wherein thegenes are selected from IpxA, IpxB and/or IpxC, or any combinationthereof.4. The Acinetobacter cell according to any one of the preceding claims,wherein the cell is obtained by deletions, and/or insertions of one orvarious nucleotides in the coding sequences of the genes.5. The Acinetobacter cell according to any one of the preceding claims,wherein the cell is an attenuated Acinetobacter cell.6. A composition comprising:

-   -   a) a cell according to any one of claims 1-2, and    -   b) a nucleic acid molecule, and/or a polypeptide.        7. The composition according to the preceding claim, wherein the        nucleic acid molecule is recombinant and the polypeptide is        recombinant.        8. The composition according to any one of claims 6-7, wherein        the polypeptide is selected from:    -   a) the peptide sequence SEQ ID NO: 27 (putative ferric        siderophore receptor (A. baumannii ATCC 17978; accession number        YP_001084684)) or a fragment thereof, wherein the fragments are        biologically active fragments, and preferably selected from the        list consisting of SEQ ID NO: 1 to SEQ ID NO: 11, or any of its        combinations, or sequences having at least 85% sequence identity        with peptide sequences SEQ ID NO: 1 to SEQ ID NO: 11, and/or    -   b) the peptide sequence SEQ ID NO: 28 (putative ferric        hydroximate siderophore receptor (A. baumannii ATCC 17978;        accesión number YP_001084696)) or a fragment thereof, where the        fragments are biologically active fragments, and preferably        selected from the list consisting of SEQ ID NO: 12 to SEQ ID NO:        23, or any of its combinations, or sequences having at least 85%        sequence identity with peptide sequences SEQ ID NO: 12 to SEC ID        NO: 23.        9. The composition according to any one of claims 6-8, further        comprising the amino acid sequence SEQ ID NO: 28 and the amino        acid sequence SEQ ID NO: 27.        10. The composition according to any one of claims 6-9 further        comprising a fusion protein comprising at least 2, preferably 3,        more preferably 4 amino acid sequences form the list SEQ ID NO:        1 to SEQ ID NO: 23 or a variant of these sequences having at        least 85% sequence identity with SEQ ID NO. 1 to SEQ ID NO: 23.        11. The composition according to claim 10, wherein the fusion        protein further comprises the amino acid sequence SEQ ID NO: 24        or the amino acid sequence SEQ ID NO: 25.        12. The composition according to any one of claims 6-11, wherein        the composition comprises a nucleotide sequence capable of        transcribing an amino acid sequence as described in any one of        claims 8-11.        13. The composition according to claim 12, wherein the        nucleotide sequence is the SEQ ID NO: 26.        14. The composition according to any one of claims 6-13, further        comprising an expression vector comprising the nucleotide        sequence according to any one of claims 12-13        15. The composition according to any one of claims 6-14, further        comprising outer membrane vesicles deficient in LPS, or cells        according to any one of claims 1 to 5.        16. The composition according to any one of claims 6-14, further        comprising at least one of the purified outer membrane proteins        of A. baumannii with amino acid sequence SEQ ID NO: 29, SEQ ID        NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO:        34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38,        SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ        ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46; SEQ ID        NO: 47; SEQ ID NO: 48; SEQ ID NO: 49; SEQ ID NO: 50; SEQ ID NO:        51; SEQ ID NO: 52; SEQ ID NO: 53; SEQ ID NO: 54; SEQ ID NO: 55;        SEQ ID NO: 56; SEQ ID NO: 57; SEQ ID NO: 58; SEQ ID NO: 59; SEQ        ID NO: 60; SEQ ID NO: 61; SEQ ID NO: 62; SEQ ID NO: 63; SEQ ID        NO: 64; SEQ ID NO: 65; SEQ ID NO: 66; SEQ ID NO: 67; SEQ ID NO:        68; SEQ ID NO: 69; SEQ ID NO: 70; SEQ ID NO: 71; SEQ ID NO: 72;        SEQ ID NO: 73; SEQ ID NO: 74; SEQ ID NO: 75; SEQ ID NO: 76, SEQ        ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID        NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO:        85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89,        or combinations thereof.        17. An outer membrane vesicle that is deficient in LPS.        18. The outer membrane vesicle according to claim 17, obtained        from a cell according to any one of claims 1-5.        19. The composition according to any one of claims 6-15, wherein        the cells or the outer membrane vesicles are designed to produce        the amino acid sequences according to any one of claim 8-11 or        16 and/or comprising the nucleotides or nucleic acids according        to any one of claims 12-13.        20. A nucleotide sequence capable of transcribing any fusion        protein according to any one of claims 10-11.        21. An expression vector comprising a nucleotide or a nucleic        acid according to any one of claims 12-13.        22. The composition according to any one of claims 7 to 16 and        19 that is a pharmaceutical composition.        23. The composition according to claim 22, further comprising a        pharmaceutically acceptable vehicle.        24. The composition according to any one of claim 22 or 23,        further comprising another active ingredient.        25. The composition according to any one of claims 22-24,        further comprising an adjuvant.        26. The composition according to any one of claims 7 to 16, 19        to 22 and 25, wherein the composition is a vaccine.        27. The composition according to any one of claims 7 to 16, 19        to 22, and 25 for use as a medicament.        28. The composition according to any one of claims 7 to 16, 19        to 22, and 25 for the prevention, improvement or the treatment        of an infection caused by A. baumannii in un mammal.        29. The composition according to any one of claims 7 to 16, 19        to 22, and 25 for conferring protection against infection caused        by A. baumannii in a mammal.        30. An antibody or active fragment thereof obtained by        immunization of a mammal with the composition according to any        one of claims 7 to 16, 19 to 22 and 25.        31. The antibody or fragment thereof of the previous claim,        wherein the composition is a pharmaceutical composition and        wherein said composition is used in therapy, particularly for        the treatment and prevention of infection caused by A.        baumannii.

ADDITIONAL CLAUSES

1.—An Acinetobacter cell deficient in LPS obtained by partial orcomplete inactivation of one or various cellular nucleic acid moleculesthat encode endogenous LPS biosynthesis genes.2.—The Acinetobacter cell according to the preceding claim, wherein thegenes are selected from IpxA, IpxB and/or IpxC, or any of theircombinations.3.—A composition comprising:

-   -   a) a cell according to any one of claims 1-2, and    -   b) a recombinant nucleic acid molecule, and/or a recombinant        polypeptide.        4.—The composition according to claim 3, wherein the polypeptide        is selected from:

c) the peptide sequence SEQ ID NO: 27 (putative ferric siderophorereceptor (A. baumannii ATCC 17978; accession number YP_001084684)) or afragment thereof, wherein the fragments are biologically activefragments, and preferably selected from the list consisting of SEQ IDNO: 1 to SEQ ID NO: 11, or any of its combinations, or sequences havingat least 85% sequence identity with peptide sequences SEQ ID NO: 1 toSEQ ID NO: 11, and/or

-   -   d) the peptide sequence SEQ ID NO: 28 (putative ferric        hydroximate siderophore receptor (A. baumannii ATCC 17978;        accesión number YP_001084696)) or a fragment thereof, where the        fragments are biologically active fragments, and preferably        selected from the list consisting of SEQ ID NO: 12 to SEQ ID NO:        23, or any of its combinations, or sequences having at least 85%        sequence identity with peptide sequences SEQ ID NO: 12 to SEC ID        NO: 23.        5.—The composition according to any one of claims 3-4, wherein        the composition comprises a nucleotide sequence capable of        transcribing an amino acid sequence described in claim 4,        preferably SEQ ID NO: 26.        6.—The composition according to any one of claims 3-5, further        comprising an outer membrane vesicle deficient in LPS, or cells        according to any one of claims 1 and 2.        7.—The composition according to any one of claims 3-6, further        comprising at least one of the proteins purified from the outer        membrane of A. baumannii SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID        NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO:        35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39,        SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ        ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46; SEQ ID NO: 47; SEQ ID        NO: 48; SEQ ID NO: 49; SEQ ID NO: 50; SEQ ID NO: 51; SEQ ID NO:        52; SEQ ID NO: 53; SEQ ID NO: 54; SEQ ID NO: 55; SEQ ID NO: 56;        SEQ ID NO: 57; SEQ ID NO: 58; SEQ ID NO: 59; SEQ ID NO: 60; SEQ        ID NO: 61; SEQ ID NO: 62; SEQ ID NO: 63; SEQ ID NO: 64; SEQ ID        NO: 65; SEQ ID NO: 66; SEQ ID NO: 67; SEQ ID NO: 68; SEQ ID NO:        69; SEQ ID NO: 70; SEQ ID NO: 71; SEQ ID NO: 72; SEQ ID NO: 73;        SEQ ID NO: 74; SEQ ID NO: 75; SEQ ID NO: 76, SEQ ID NO: 77, SEQ        ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID        NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO:        86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, or any        combination thereof.        8. An outer membrane vesicle deficient in LPS.        9. The outer membrane vesicle according to claim 8, obtained        from a cell as described in any one of claims 1-2.        10. The composition according to any one of claims 3-7, wherein        the cells or the outer membrane vesicles are designed to produce        the polypeptide sequences according to any one of claims 4 and        7, and/or comprising the nucleic acid sequences of claim 5.        11. The composition according to any one of claims 3-7 and 10,        wherein the composition is a pharmaceutical composition.        12. The composition according to any one of claims 3-7, 10 and        11 for use as a medicament.        13. The composition according to any one of claims 3-7, 10 and        11 for use in the prevention, improvement or treatment of an        infection caused by A. baumannii in a mammal.        14. An antibody or active fragment thereof obtained by        immunization of a mammal with the composition according to any        one of claims 3 to 7, 10 and 11.        15. The antibody or the active fragment thereof according to the        preceding claim, wherein the composition is a pharmaceutical        composition, and where said composition is used in therapy,        particularly for the treatment or prevention of an infection        caused by A. baumannii.

1.-17. (canceled)
 18. A method of producing an antibody or fragment thereof that targets Acinetobacter baumannii, comprising: a) contacting an antibody or fragment thereof or an antibody or antibody fragment library with an A. baumannii strain deficient in lipopolysaccharide (LPS) and/or an outer membrane vesicle (OMV) derived therefrom; and b) selecting those antibodies or fragments thereof having affinity or binding affinity or capable of specifically binding the A. baumannii strain and/or OMV derived therefrom.
 19. The method of claim 18, further comprising isolating the antibody or fragment thereof identified in step b) above.
 20. The method of claim 18, wherein the antibody or fragment thereof is a Fab, Fab′, Fab′-SH, Fv, scFv, F(ab′)2, Vhh, Nanobody, or diabody.
 21. The method of claim 18, wherein the A. baumannii strain deficient in LPS is characterized by the partial or complete inactivation of one or more genes selected from the group consisting of lpxA, lpxC, and lpxD.
 22. The method of claim 18, wherein the A. baumannii strain deficient in LPS is derived from ATCC strain
 19606. 23. The method of claim 18, wherein the A. baumannii strain deficient in LPS is an ATCC 19606 strain with a mutation in one or more genes selected from the group consisting of lpxA, lpxC and lpxD.
 24. A method of producing an antibody or fragment thereof that targets Acinetobacter baumannii, comprising: administering an A. baumannii strain deficient in lipopolysaccharide (LPS) and/or an outer membrane vesicle (OMV) derived therefrom to a mammal.
 25. The method of claim 24, further comprising isolating the antibody or fragment thereof.
 26. The method of claim 24, wherein the antibody or fragment thereof is a Fab, Fab′, Fab′-SH, Fv, scFv, F(ab′)₂, Vhh, Nanobody, or diabody.
 27. The method of claim 24, wherein the A. baumannii strain deficient in LPS is characterized by the partial or complete inactivation of one or more genes selected from the group consisting of lpxA, lpxC, and lpxD.
 28. The method of claim 24, wherein the A. baumannii strain deficient in LPS is derived from ATCC strain
 19606. 29. The method of claim 24, wherein the A. baumannii strain deficient in LPS is an ATCC 19606 strain with a mutation in one or more genes selected from the group consisting of lpxA, lpxC and lpxD. 